Variants of alpha-fetoprotein coding and expression sequences

ABSTRACT

The invention discloses the sequences of variant forms of alpha-fetoprotein transcripts that have been identified in human hemopoietic progenitors but not in differentiated mature cells. The variant forms of AFP (vAFP) cDNA sequences isolated from a multipotent hemopoietic cell line, K562, differ from the authentic AFP transcript, consisting of 15 exons, by lacking only exon 1. Instead of exon 1, vAFP transcripts use an additional one or two exons located in the 5′-untranslated region of the AFP gene. K562 expressed selectively vAFP, whereas a hepatocellular carcinoma cell line, HepG2, showed no detectable expression of vAFP. In normal adult tissues, vAFP transcripts is detected in the bone marrow, thymus and brain, but not the spleen, suggesting the expression occurs in normal hemopoietic progenitors. Moreover, CD34+Lin− hemopoietic stem/progenitor cells purified by flow cytometric sorting also express the variant transcripts.

1.0 CROSS REFERENCE TO RELATED APPLICATION DATA

This application claims priority to U.S. Provisional Application60/324,540, filed Sep. 26, 2001, the disclosure of which is herebyincorporated by reference in its entirety.

2.0 FIELD OF THE INVENTION

The present invention relates generally to methods of identifying formsof alpha-fetoprotein unique to hemopoietic progenitors and tohemopoietic cancers and thereby, hemopoietic stem cells or progenitorsand cancers related thereto, as a marker for cell cloning andidentification of cloned cells, and for evaluating developmental stagesin organs and organisms.

3.0 BACKGROUND OF THE INVENTION

Multipotent stem cells, cells capable of extensive growth without losingtheir potential for differentiating into a plurality of cell types, arefound in fetal and various adult tissues. Multipotent stem cells havebeen isolated from fetal tissue and bone marrow.

Cell differentiation in the developing embryo is regulated by extrinsicinductive signals and an intrinsic programmed genetic code.Differentiation into the three germ layers (ectoderm, endoderm, andmesoderm) from primitive ectoderm (epiblast) is a crucial step duringdevelopment and previously thought to be an irreversible process leadingto a germ layer giving rise to unique types of cells, e.g., epidermal orneuronal cells from ectoderm; epithelial cells in internal organs ordigestive tract from endoderm; and hemopoietic and mesenchymal cellsfrom mesoderm. Recent studies of cell transplantation, however, haveindicated that somatic stem cells or progenitor cells from adult tissuesand with characteristic tissue-specific markers were able, possibly, togenerate cells with fates different from those heretofore recognized asdescendants of a specific germ layer. Although the evidence for this isstill inconclusive, if proven true it would an example of a processcalled “transdifferentiation” and leads to questions about possiblemechanisms. A candidate example of putative transdifferentiation is thatof CD45+ hemopoietic stem cells giving rise to mature hepatocytes.

Adult liver parenchymal cells consist of hepatocytes and biliaryepithelial cells. They are derived from common precursors, hepatoblasts,that come from foregut endodermal stem cells by an inductive signal(s)from the septum transversum surrounding the outpouching of the endoderm.Although it is not known whether hemopoietic cells can differentiateinto hepatoblasts, there does appear to be a subpopulation ofprogenitors in the bone marrow capable of maturing into hepatocytes andsharing critical antigenic markers with that of hemopoietic progenitors;therefore, these hemopoietic progenitor cells should express someendodermal markers before full differentiation into hepatocytes.

Alpha-fetoprotein (AFP) is a major serum protein produced primarily byendoderm-derived yolk sac and by hepatoblasts as well as moredifferentiated fetal hepatic cells. AFP is one of the earliest markersfor endodermal differentiation; the transcriptional expression starts atvisceral and definitive endoderm in the early embryo and is regulatedtightly in a developmental and tissue-specific manner. Therefore, inmost studies in which is assessed endodermal differentiation of humanembryonic stem cells or embryonic germ cells, the expression of AFP mRNAhas been investigated and used as a marker of endoderm.

4.0 SUMMARY OF THE INVENTION

The inventors have identified at least two variant forms of human AFPtranscripts. The variant forms of human AFP transcripts are associatedwith certain non-hepatic tissues. In particular, the variant forms areassociated with a multipotent hemopoietic cell line, K562, and with bonemarrow progenitors. The cDNA sequences revealed that the differences inthe variant AFP (vAFP) mRNAs compared to that of the authentictranscript, consisting of 15 exons, are the presence of one or twounique exons, named exon A and exon B, replacing exon 1 of AFP. Thevariant forms were detected in normal CD34⁺ Lin⁻ hemopoietic progenitorcells but not in mature blood cells. The expression of the variant AFPtranscripts suggests that hemopoietic progenitors are in an immaturestate that is permissive to express certain types of transcripts thathave been considered unique to endoderm.

Accordingly, one aspect of the invention is an isolated nucleic acidhaving a polynucleotide sequence corresponding to SEQ ID NO:1, a homologthereof, or a complement thereof. The nucleic acid can be suitable fordetection of a variant form of alpha-fetoprotein mRNA.

One aspect of the invention is a nucleic acid primer in which the primercomprises at least ten contiguous nucleotides in the polynucleotidesequence corresponding to SEQ ID NO:1.

In one aspect, the invention comprises a polypeptide translated from anucleic acid comprised at least in part by a polynucleotide sequencecorresponding to SEQ ID NO:1 or a complement thereof. In one embodimentthe polynucleotide sequence is at least 97% homologous to SEQ ID NO:1.

One aspect of the invention is an isolated nucleic acid having apolynucleotide sequence corresponding to SEQ ID NO:2, a homolog thereof,or a complement thereof. In one embodiment the nucleic acid is suitablefor detection of a variant form of alpha-fetoprotein mRNA.

In one aspect, the invention is a nucleic acid primer in which theprimer comprises at least ten contiguous nucleotides of a polynucleotidesequence corresponding to SEQ ID NO:2.

One aspect of the invention is a polypeptide translated from a nucleicacid comprised at least in part by a polynucleotide sequencecorresponding to SEQ ID NO:2. In one embodiment the polynucleotidesequence is at least 97% homologous to SEQ ID NO:2.

In one aspect, the invention is a composition suitable for detection ofvariant alpha-fetoprotein mRNA comprising: (a) a first nucleic acidprimer in which the first primer comprises at least ten contiguousnucleotides of the polynucleotide sequence corresponding to SEQ ID NO:1,and optionally (b) a second nucleic acid primer in which the secondprimer comprises at least ten contiguous nucleotides of thepolynucleotide sequence corresponding to SEQ ID NO:2.

One aspect of the invention is a composition comprising a nucleic acidin which the nucleic acid encodes a polypeptide encoded in all or inpart by the nucleic acid of SEQ ID NO:1 or the nucleic acid of SEQ IDNO:2. In another embodiment the invention is a composition comprising anisolated nucleic acid having a polynucleotide sequence corresponding toSEQ ID NO:1., SEQ ID NO:2, or complements thereof.

In one aspect, the invention is a nucleic acid with a sequence spanningthe splice from exon A to exon 2, from exon A to exonB, or from exon Bto exon 2. Thus, in one aspect, the invention is a composition suitablefor detection of a variant form of alpha-fetoprotein, the compositioncomprising a polynucleotide of sequence N1-C-C-A-A-G-C-T-T-N2 (SEQ IDNO:67), N1-C-C-A-A-G-G-T-A-N2 (SEQ ID NO:68), or N1-G-G-A-G-A-C-T-T-N2(SEQ ID NO:69), in which each of N1 and N2 have a sequence selected fromthe group consisting of AA, AC, AG, AT, AX, CA, CC, CG, CT, CX, GA, GC,GG, GT, GX, TA, TC, TG, TT, TX, XA, XC, XG, XT, and XX, in which A, C,G, T, and X represent the 2′-deoxyribonucleic acid moieties of adenine,cytidine, guanine, thymidine, and no nucleic acid, respectively.

One aspect of the invention is a composition suitable for detection of avariant alpha-fetoprotein comprising a polynucleotide of sequenceN1-N2-N3-N4-N5-N6-N7-N8-N8-N10, in which each of N1, N2, N3, N4, N5, N6,N7, N8, N9, and N10 are independently A, C, G, T, or no nucleotidemoiety, with the proviso that no more than one nucleotide can vary fromthe sequence C-C-C-A-A-G-C-T-T-C (SEQ ID NO:70), C-C-C-A-A-G-G-T-A-T(SEQ ID NO:71), or T-G-G-A-G-A-C-T-T-C (SEQ ID NO:72). In thisembodiment, the length of the polynucleotide can be from nine to 25bases, for example ten, or twelve.

In one aspect, the invention is a polynucleotide primer comprising:seven or more nucleotide residues capable of hybridizing under stringenthybridization conditions to a nucleic acid encoding a variant form ofalpha-fetoprotein. In one embodiment the nucleic acid is not capable ofhybridizing to a second nucleic acid encoding normal, hepatic-specificalpha-fetoprotein.

The stringent hybridization conditions comprise any conditionsconsidered stringent in the art of the invention. In one embodimentstringent hybridization conditions comprise 3× to 8×SSC. In anotherembodiment the hybridization conditions comprise 6×SSC, 0.5% SDS at 65°C., and washing at 2×SSC at room temperature. In another embodiment thehybridization conditions are 7% (w/v) sodium dodecylsulfate, 0.5M NaPO₄,pH 7.0, 1 mm EDTA, at 50° C.; followed by washing with 1% sodiumdodecylsulfate.

In one aspect, hybridization is carried out at 55-65° C. underconditions of 6×SSC, 0.5% SDS, and washing at 2×SSC at room temperature.

One aspect of the invention is a method of detecting a variantalpha-fetoprotein mRNA comprising: (a) combining a sample suspected ofcontaining a variant form of alpha-fetoprotein, at least one primercapable of hybridizing to a variant form of AFP mRNA, and reagents forPCR to form a mixture, (b) subjecting the mixture to thermocycling, and(c) determining the absence or presence of cDNA corresponding to avariant form of AFP mRNA. In one embodiment the primer comprises atleast ten continguous nucleotides of a polynucleotide sequencecorresponding to SEQ ID NO:1, SEQ ID NO:2, or complements thereof. Inone embodiment the mixture comprises a first primer comprising at leastten contiguous nucleotides corresponding to SEQ ID NO:1 or a complementthereof and a second primer comprising at least ten contiguousnucleotides of a polynucleotide sequence corresponding to SEQ ID NO:2 ora complement thereof. In another embodiment the mixture comprises afirst primer comprising at least ten contiguous nucleotidescorresponding to SEQ ID NO:1, SEQ ID NO:2, or complements thereof and asecond primer, comprising at least ten contiguous nucleotides of apolynucleotide sequence corresponding to mammalian AFP, or a complementthereof. In one embodiment the primers can be between about 15 and about25 nucleotides in length.

One aspect of the invention is a method of identifying or detectinghemopoietic stem or progenitor cells comprising determining the presenceor absence of a variant form of alpha-fetoprotein mRNA in cellssuspected of being hemopoietic stem or progenitor cells.

In one aspect, the invention is an isolated nucleic acid encoding avariant form of alpha-fetoprotein (AFP) preferentially expressed inprogenitor cells, in which exon 1 of exons 1-14 of alpha-fetoprotein hasbeen replaced by a polynucleotide sequence corresponding to SEQ ID NO:1,SEQ ID NO:2, or a fusion thereof.

One aspect of the invention is a probe capable of detecting theexpression of a variant form of alpha-fetoprotein (AFP) comprising anucleic acid consisting essentially of at least 10 contiguousnucleotides of the polynucleotide sequence corresponding to SEQ ID NO:1,a homolog thereof, or a complement thereof.

In one aspect, the invention is a recombinant expression vectorcomprising the isolated nucleic acid of SEQ ID NO:1, SEQ ID NO:2, orboth.

One aspect of the invention is a method for detecting a polynucleotide,which encodes a variant form of AFP, in a sample comprising: (a)hybridizing the nucleic acid corresponding to either SEQ ID NO:1 or aportion thereof having 10 or more nucleotides, to a nucleic acid presentin a sample to be tested to form a hybridization complex; and (b)detecting the hybridization complex, if any. The presence of thehybridization complex can indicate the presence of a polynucleotideencoding a variant form of AFP in the sample. Moreover, the conditionsfor hybridization can be stringent.

In one aspect, the invention is a method of preparing a subpopulation ofcells enriched in hemopoietic progenitors comprising: (a) providing acell suspension comprising subpopulations of various types of cells and(b) selecting a subpopulation of cells which expresses a variant form ofalpha-fetoprotein nucleic acid to provide an enriched population ofhemopoietic progenitors is provided. In another aspect, the invention isa composition comprising cells capable of expressing vAFP, at least aportion of which cells are hemopoietic progenitors, or their progeny.The cells, or at least a subpopulation thereof, can be from bone marrow.In one aspect, the invention is a composition comprising at least onesubpopulation of cells which comprises hemopoietic progenitors, or theirprogeny, capable of expressing variant AFP. The variant AFP can includeexon A, exon B, combinations thereof, or variants thereof Thesubpopulation of the composition can optionally be derived from bonemarrow.

One aspect of the invention is a method of identifying a tumorcomprising:

-   -   (a) providing a tumor or a tissue sample suspected of including        tumor tissue and    -   (b) detecting the absence or presence of variant        alpha-fetoprotein nucleic acid whereby a tumor is identified.

In one aspect, the invention is a method of identifying a hemopoieticprogenitor comprising: (a) providing a putative progenitor and (b)detecting a variant alpha-fetoprotein nucleic acid whereby a hemopoieticprogenitor is identified.

One aspect of the invention is a method of identifying a hepatopoieticprogenitor comprising: (a) providing a putative progenitor and (b)measuring a substantial absence of variant alpha-fetoprotein nucleicacid whereby a hepatopoietic progenitor is identified.

One aspect of the invention is a polypeptide having an amino acidsequence corresponding to SEQ ID NO:17 or an analog thereof. Oneembodiment of the invention is an antibody against a polypeptide havingan amino acid sequence corresponding to SEQ ID NO:17, or an analogthereof, or a fragment thereof. In one embodiment the fragment is atleast about three amino acids and in another embodiment the fragment isat least about four amino acids.

5.0 BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 panel A depicts the exon structure of the AFP genome and panel Bdepicts the splicing of exons to form AFP mRNA.

FIG. 2 panel A illustrates sizing gel electropherograms of mRNA exonsidentified by rt-PCR and panel B illustrates the identification of exonsused in AFP transcripts.

FIG. 3 panel A depicts the nucleotide sequences of exons A, B, 2, and 3(SEQ ID NOS :37-39 disclosed respectively in order of appearance) andpanel B depicts the splicing of mRNA exon transcription products to formnormal AFP (top), variant AFP-A (middle), and variant AFP-B (bottom).

FIG. 4 depicts the genomic sequence of variant AFP exon A (SEQ IDNO:40).

FIG. 5 depicts selective expression of variant forms of AFP in K562cells.

FIG. 6 depicts selective expression of variant AFP transcripts in normalhuman tissues. Panel A depicts the splicing of exons to form the mRNA oftwo variants of AFP, and panel B depicts sizing gel electrophereogramsof rt-PCT of variant AFP (top), normal AFP (second from top), beta actin(third from top), and glycerol-3-phosphate dehydrogenase (bottom).

FIG. 7 panel A depicts a two color FACS sort and panel B depicts sizinggel electropherograms of variant AFP, CD34, and beta-actin.

6.0 DETAILED DESCRIPTION OF THE INVENTION

In one aspect, the invention is a method of assay for a variantalpha-fetoprotein mRNA comprising: (a) combining a sample, a firstprimer, a second primer, and reagents for PCR to form a mixture, (b)subjecting the mixture to thermocycling, and (c) identifying the absenceor presence of variant AFP cDNA, in which the first primer, the secondprimer, or both are capable of hybridizing to the variantalpha-fetoprotein mRNA. This method can be one in which the first primercomprises at least ten contiguous nucleotides according to the DNA ofSEQ ID NO:1 or a complement thereof. In one aspect, this method can beone in which the second primer comprises at least ten contiguousnucleotides according to the DNA of SEQ ID NO:2 or a complement thereof.In another alternative, the second primer comprises at least tencontiguous nucleotides according the DNA sequence of a mammalian AFP, ora complement thereof. In one embodiment the mammal is human.

One aspect of the invention is a method of identifying or detectinghemopoietic stem or progenitor cells comprising determining the presenceor absence of a variant form of alpha-fetoprotein mRNA in cells or asample from cells, suspected of being hemopoietic stem or progenitorcells. This method can be one in which the determining step comprisesPCR using one or more primers to one or more variant forms ofalpha-fetoprotein. This method can also be one in which the determiningstep comprises immunodetection, for example using antibody to at least apart of the variant alpha-fetoprotein. The immunodetection can be anymethod standard in the art, including ELISA, RIA, immunohisto chemistryand, immunofluorescence. The antibody can be directed to any epitope ofa variant form of AFP.

In one aspect, the invention is an isolated nucleic acid encoding avariant form of AFP preferentially expressed in progenitor cells, inwhich exon 1 of exons 1-14 of AFP has been replaced by a polynucleotidesequence corresponding to SEQ ID NO:1, SEQ ID NO:2 or a fusion thereof.In one embodiment, the nucleic acid includes at least 100 contiguousnucleic acid residues corresponding to the N-terminal half of a variantform of AFP. In another embodiment, the nucleic acid includes at least50 contiguous nucleic acid residues corresponding to the N-terminal halfof a variant form of AFP.

One aspect of the invention is a probe for detection, measurement, orboth, of a AFP variant gene expression, which comprises any one of (a) apurified, isolated or synthesized DNA consisting of a sequence of atleast 10 contiguous nucleotides in the DNA of SEQ ID NO:1, (b) apurified, isolated or synthesized DNA complementary and identical inlength to DNA (a), or (c) a purified, isolated or synthesized DNA havingat least 90% homology to DNA (a) or (b); or a polynucleotide sequencewhich is complementary thereto. In one embodiment the homology is about99%. The probe can be of any length standard in the art without limitingto a specific length, the probe can be 100 to 300 nucleotides in length.The probe can be about 150 to about 250 nucleotides in length.

One aspect of the invention is a method for detecting a polynucleotidewhich encodes a variant form of AFP in a sample comprising: (a)hybridizing the nucleic acid corresponding to either SEQ ID NO:1 or SEQID NO:2, or a portion thereof having 10 or more nucleotides, to anucleic acid in the sample to be tested to, form a hybridizationcomplex; and (b) detecting the hybridization complex, if any. Thepresence of the hybridization complex indicates the presence of apolynucleotide encoding variant AFP in the sample. In this method thenucleic acid can be amplified by the polymerase chain reaction prior tohybridization.

Stringent hybridization conditions are those known in the art thatpermit hybridization of only closely homologous polynucleotides. Thestringent conditions can include a hybridization medium having 6×SSC,0.5% SDS, used at a temperature of 65° C. or any other medium andcondition known in that art as a stringent condition. Hybridization canbe followed by washing in 2×SSC at room temperature.

The invention can be illustrated further by the figures provided.

FIG. 1 is a schematic representation of the human AFP gene, the AFPtranscript, and the position of primers used for RT-PCR. Panel Aillustrates that the Human AFP gene consists of 15 exons and 14 intronsand spans approximately 20,000 base pairs. Part B illustrates thelocation of the initiation methionine codon (ATG) and the terminationcodon (TAA) of a AFP transcript in exon 1 and exon 14, respectively. Theapproximate positions of sense primers and anti-sense primers are shown.The nucleotide sequences of primers are described in Table 1.

FIG. 2 depicts the expression of a variant form of AFP mRNA in the cellline K562, a hematopoietic cell line. In panel A, the expression of AFPmRNAs in K562, and hepatocellular carcinoma cell line, HepG2, areanalyzed by RT-PCR using three different primer combinations. Primercombinations of ex-1S and ex-3A, ex-12S and ex-14A, and ex-1S and ex-14Aare used to amplify exon 1 to exon 3 (lane 1, 4, and 7), exon 12 to exon14 (lane 2, 5, and 8), and exon 1 to exon 14 (lane 3, 6, and 9),respectively. Control reaction is performed without a template. TheC-terminus part of AFP transcript is expressed in K562. Panel B depictsthe identification of exons used in a AFP transcript from K562. RT-PCRof primer combinations of a series of 5′ primers from exon 1 to exon 6(ex-1S to ex-6S) and ex-14A as the 3′ primer are performed with cDNAfrom K562 (lane K) and HepG2 (lane H). HepG2 cDNA is diluted at one toone hundred times. K562 expresses the entire coding exons, except forexon 1, in the authentic AFP transcript. The right sections of panels Aand B show expression of beta-actin (A: lane 10 HepG2; lane 11, K562;lane 12, control and B: lane 15, control). The open arrow heads indicate1 k bp.

FIG. 3 depicts sequences and the genomic structure of two variant formsof AFP expressed in K562. Panel A depicts DNA sequences of two variantforms of AFP transcripts isolated from K562 cDNA. Panel B is a schematicillustration of the genomic structure of a variant form of AFP. Openboxes and horizontal lines indicate exons and introns, respectively.Their lengths in nucleotides are indicated by numbers above and belowthese elements. One type of the variant forms used one additional exon(exon A) located at approximately 5 kb upstream from exon 1. The othertype used exon A and another exon (exon B) located at approximately 1.6kb from exon 1. The genomic organization of the variant forms wasdeduced from the genomic sequence of the AFP gene.

FIG. 4 depicts the genomic sequence of a variant form of AFP exon A. Thenucleotide sequence in the open box indicates exon A cloned by anchoredPCR. The nucleotide position from the top left is indicated on theright. The sequence between 517 to 739 is confirmed as present in K562,HepG2, and MRC5. Open circles mark nucleotides which differ from thosein a previous report in which the nucleotides indicated underneath.Facing arrows and a double underline indicate an inverted repeat andputative TATA box, respectively. Possible binding sites oftranscriptional factors GATA-X (closed circles), MZF-1 (open squares),AML-1 (closed squares), and NF-Y (closed triangles) are shown abovenucleotides. Binding motifs are analyzed by TRANSFAC.

FIG. 5 depicts selective expression of variant AFP transcripts in K562.RT-PCR of the primer combination of exon A primer (ex-AS) and ex-14Aprimer is performed with cDNA from K562 (lane 1) and HepG2 (lane 2).K562, but not HepG2, expresses a variant form of an AFP transcript. Notethe strong signal of HepG2 by RT-PCR using ex-2S and ex-14A. Thisindicates that HepG2 expresses the authentic form only. Equal amounts ofcDNA are used for the reaction. Lane 3 is a control reaction.

FIG. 6 depicts RT-PCR analyses of variant forms of AFP transcripts innormal human tissues. Panel A depicts a nested PCR strategy. A first PCR(PCR-1) of the primer combination of ex-A1S/ex-14A and a second PCR(PCR-2) of that of ex-A2/ex-3A are illustrated. The amplified DNAfragments of two AFP variant forms are distinguishable in agaroseelectrophoresis. Panel B depicts analysis of the expression of variantAFP mRNAs in cDNAs from various human tissues: lane 1, bone marrow; lane2, thymus; lane 3, spleen; lane 4; small intestine; lane 5, colon; lane6, stomach; lane 7, brain; lane 8, heart; lane 9, kidney; lane 10,liver; lane 11, lung; lane 12, trachea; lane 13, MRC5; lane 14, K562;and lane 15, no template. Nested RT-PCR of authentic AFP transcript isperformed in the same human tissues cDNA. The results of single stepRT-PCR of beta actin and G3PDH are shown. Expression of variant AFPmRNAs is detected in bone marrow, thymus, and brain. Open and closedarrowheads indicate 500 bp of 100 bp ladder and 1000 bp of 1000 bpladder marker, respectively.

FIG. 7 depicts expression of variant forms of AFP transcripts in normalhuman hematopoietic progenitors. Panel A depicts representative dotplots of flow cytometric analysis for CD34 and CD38 expression inumbilical cord blood cells after ficoll centrifugation (left). The CD34+cell population on the average is 2.3% of the whole. The CD34+ cellpopulation is enriched after removing Lin+ cells (right). The live CD34cell fraction (inside of open box) is sorted for RT-PCR analysis. PanelB depicts RT-PCR analysis of variant forms of AFP transcripts in sortedumbilical cord blood cells. Roman numerals indicate individual UCBsamples. RNAs are isolated from unfractionated (lane 1, 3, 5, and 7) orCD34+Lin− UCB cells (lane 2, 4, 6, and 8), and nested PCR is performedas described in FIG. 5A. CD34+Lin− cells in UCB expressed variant formsof AFP. Lane 9 is K562 cells and lane 10 is a control reaction.

7.0 EXAMPLES

The following examples are illustrative of specific uses of theinvention, but the claimed invention is not limited by the specificexamples provided.

7.1 Variant AFP mRNA Expressed in K562, a Human Erythroleukemia CellLine

The human AFP gene consists of 15 exons, in which the coding sequence isfrom exon 1 to exon 14 (FIG. 1A). Two different portions of the AFP cDNAsequence are selected as target sequences of RT-PCR. The primercombination of ex-1S and ex-3A is for the amplification of exon 1containing the initiation methionine to exon 3, whereas that of ex-12Sand ex-14A amplifies exon 12 to exon 14 containing the termination codon(FIG. 1B). The results of the PCR amplification are shown in FIG. 2A.Both combinations of the primers resulted in amplification bands whichare strongly detected in the RNA from HepG2, a hepatocellular carcinomacell line. By contrast, only the specific band of the C-terminal portionis detected by the primer set of ex-12S and ex-14A in the RNA from K562,an erythroleukemia cell line. These results might suggest that K562expresses a short form of the authentic AFP (authentic AFP) transcriptwithout the N-terminus. In fact, the result of the PCR for the wholecoding region of AFP using ex-1S and ex-14A primers shows that thesingle remarkable band of 1.8 Kb (lane 3) is amplified from the HepG2cDNA, whereas there is no band in K562 (lane 6).

Only one AFP gene per haploid genome has been demonstrated in rats,mice, and humans. In all three species, the AFP genes are organizedsimilarly into 15 coding exons interrupted by 14 introns. Although thereis no report about any variant form of the AFP mRNA in humans, severalshort forms of the AFP transcript have been characterized in rat. Allthose transcripts share a common 3′ sequence. Detailed analysis of onevariant form of them showed that the rat vAFP lacks the first sevenexons of authentic AFP mRNA. Thus, the inventors designed new senseprimers for exon 7 and exon 8 to see whether the variant form of humanAFP mRNA in K562 is similar to those of rat, because a designated V exonlocated in the seventh intron of the rat gene has been identified as thefirst exon of the variant forms. By RT-PCR analysis, the primer for exon7, as well as one for exon 8, detected vAFP in K562. Thus, the humanvAFP transcript is not similar to that in the rat. Therefore, a seriesof 5′ primers from exon 2 to exon 6 are constructed (FIG. 1) to detectthe difference between authentic and variant forms of AFP transcripts.Surprisingly, the entire coding exons, except for exon 1, are shared inthe variant form of AFP in K562 (FIG. 2 b).

7.2 Molecular Cloning of Variant AFP cDNA from K562

To identify the structure of the N-terminus portion of the vAFPtranscript in K562, anchored PCR is performed by standard methods. As aresult, two types of variant transcripts are identified, as shown inFIG. 3A. Comparing the sequences of the variant transcripts to theGenbank database, two regions of genomic sequence of human AFP gene areidentified. One type of the variant forms uses an additional exon,designated exon A, located at 5 kb upstream from exon 1 (FIG. 3B). Theother type used exon A and another exon, designated exon B, located at1.6 kb from exon 1 (FIG. 3B). Among 19 clones analyzed, 15 clones aretype-A; 3 clones are type-AB; and one clone is the germ line transcriptof intron 1. Although the genomic sequences of AFP region were reportedfrom a couple of sources, some nucleotides in the exon A of K562 differfrom the reported genomic sequence by one nucleotide base. Therefore,the K562 genomic sequence of exon A is compared to the cloned cDNAsequence to determine whether the difference results from artifacts ofthe anchored PCR. While the variant cDNA and genomic DNA of K562 areidentical, a TC-rich sequence at the right upstream of the varianttranscripts in K562 is substituted to produce the AC-rich sequence inthe previous report. Thus, the genomic sequence of normal humanfibroblasts as well as HepG2 is compared to that of K562 to see whetherthe mutation is K562-specific or not. However, no differences in allthree genomic sequences of the exon A region of K562 are observed. InFIG. 4 is shown the overall genomic sequence in the region of exon Afrom our data combined with the Genbank database. As shown in thefigure, possible binding sites of several transcriptional factors suchas GATA-X, MZF-1, and AML-1, with functions crucially associated withearly hematopoiesis, can be identified around exon A. Also, a possibleTATA box, albeit not a typical one, is indicated. In addition, aninverted repeat of 30 nucleotides, including the TC-repeat sequence withthe 180 bp interval, is identified (FIG. 4).

7.3 Selective Expression of Variant Transcripts in K562

The expression pattern of the variant and authentic transcripts of AFPis studied in K562 and HepG2. A sense primer of exon A for RT-PCR isdesigned. The combination of the exon A primer and exon 14 primer,ex-14A, successfully detects the transcripts in K562, but not HepG2. Thedetected bands in K562 are cloned and sequenced to confirm the amplifiedproducts. As expected, DNA sequencing shows that the two types ofvariant AFP transcripts uses exon A and B or exon A only and arefollowed by exon 2 to exon 14. The number of clones with a type Asequence is 7 out of 12, while that of the type AB clone is 5 out of 12.This suggests that two types of vAFP are expressed equally in K562. Thefar stronger signal of HepG2 by RT-PCR using ex-2S and ex-14A indicatesthat HepG2 is expressed the authentic form only. This result clearlydemonstrates that the pattern of expression of variant or authenticforms of AFP in K562 is opposite to that of HepG2.

7.4 Expression of Variant Forms of AFP in Normal Human Tissues

Variant transcripts of AFP expressed in normal cells are assessed bynested (two step) RT-PCR to detect the very low level expression. Thefirst PCR is performed with primers, ex-A1S and ex-14A, to amplify wholethe coding sequence of variant forms. Subsequently, nested PCR iscarried out by an internal primer combination, ex-A2S and ex-3A. Thisnested PCR can distinguish type A and type AB by the molecular size(FIG. 5A). The results of PCR show that the tissue distribution of thevariant AFP transcripts is fairly restricted. Only brain and primaryhemopoietic organs, such as bone marrow and thymus, express thetranscripts. Other tissues, including liver, lung, trachea, kidney,stomach, small intestine, colon, heart and spleen, do not have cellsexpressing vAFP. On the other hand, authentic AFP is detected in brain,kidney, small intestine, and thymus, but not in bone marrow. The patterncorresponds with that of embryonic expression of authentic AFP. Theseresults strongly suggest that hemopoietic progenitors, but notdifferentiated cells, express vAFP, since spleen is a secondaryhemopoietic organ, in which hemato-lymphopoiesis does not normallyoccur. In addition, cDNA from peripheral blood cells does not show theexpression of vAFP. The lack of expression in normal human fetalfibroblasts (FIG. 6B, lane 13) and small intestine (FIG. 6B, lane 4)suggests that the vAFP expression is not associated simply with thestatus of cell proliferation.

7.5 Variant Transcripts of AFP Expressed in Hemopoietic Progenitors

The RT-PCR of tissue RNAs cannot define which cells express the variantforms of AFP transcripts, because tissues are comprised of many celltypes. Bone marrow and thymus, for example, consist of veryheterogeneous cell populations. Therefore, purified hemopoieticprogenitors from umbilical cord blood are evaluated by flow cytometricsorting to learn whether or not they express vAFP. CD34+CD38−Lin− cellsare a negligible subpopulation in unfractionated umbilical cord blood(FIG. 6A) but consist of pure hemopoietic progenitors and includehemopoietic stem cells. The CD34+CD38− cells are highly enriched afterremoving Lin-positive cells (FIG. 6A). Both unfractionated live cells(7AAD−) and CD34+Lin− cells are isolated by flow cytometric sorting, andthe RNAs extracted are subjected to nested PCR for vAFP. A total of fourdifferent cord blood samples are tested. As shown in FIG. 6B, theexpression of vAFP transcripts are detected successfully in all samplesof enriched hemopoietic progenitors (lanes 2, 4, 6, and 8), butgenerally not in whole cord blood cells (lanes 1, 3, and 5), althoughobserved in sample 7. The amplified bands are exactly identical DNAsequences to those of type A and type AB from K562 (FIG. 3A) by cloningand DNA sequencing. Similarly, cells are isolated from other tissues,for example bone marrow, and evaluated for expression of vAFP before andafter optional cell sorting for surface markers, for example CD45, theLin markers, or both.

7.6 A Homolog of vAFP: G¹⁰T²⁰-variantA-AFP¹⁹⁷

A homolog of a variant AFP, designated G¹⁰T²⁰-variant A-AFP¹⁹⁷, anddescribed as SEQ ID NO:3, varies from variant A-AFP by single pointsubstitutions at two loci. The G¹⁰T²⁰-variant A-AFP¹⁹⁷ and complementsthereof and portions thereof can be used for identification of variantAFP in cells—particularly in stem and progenitor cells from bone marrowand other hemopoietic organs, for cell cloning and identification ofcloned cells, for tumor identification, for evaluating developmentalstages in organs and organisms, and as a hybridization marker for exon 1or variants of exon 1. Other homologs of varA-AFP with about 99%homology are also useful for these purposes.

7.7 A Homolog of vAFP: A³⁰C⁹⁰-variantA-AFP¹⁹⁷

A homolog of variant AFP, designated A³⁰C⁹⁰-variant A-AFP¹⁹⁷, anddescribed as SEQ ID NO:4, varies from variant A-AFP by single pointsubstitutions at two loci. The A³⁰C⁹⁰-variant A-AFP¹⁹⁷ and complementsthereof and portions thereof can be used for all the purposes thatvariantA-AFP can be used. Among suitable uses for A³⁰C⁹⁰-variantA-AFP¹⁹⁷ can be (a) identification of variant AFP in cellisolation—particularly in isolation of stem and progenitor cells frombone marrow and other hemopoietic organs, (b) cell cloning andidentification of cloned cells, (c) tumor identification, (d) evaluationof developmental stages in organs and organisms, and (e) as ahybridization marker for exon 1 or variants of exon 1. Other homologs ofvarA-AFP with about 99% homology are also useful for these purposes.

7.8 A Homolog of vAFP: variantA-(des-T⁷¹)(des-A¹⁵²)AFP¹⁹⁵

A homolog of variant AFP, designated variantA-(des-T⁷¹)(des-A¹⁵²)AFP¹⁹⁵, and described as SEQ ID NO:5, varies fromvariant A-AFP by two nucleotide deletions. The variantA-(des-T⁷¹)(des-A¹⁵²) AFP¹⁹⁵ and complements thereof and portionsthereof can be used for identification of variant AFP in cellisolation—particularly in isolation of stem and progenitor cells frombone marrow and other hemopoietic organs, for cell cloning andidentification of cloned cells, for tumor identification, for evaluatingdevelopmental stages in organs and organisms, and as a hybridizationmarker for exon 1 or variants of exon 1. Other homologs of varA-AFP withabout 99% homology and having nucleotide deletions are also useful forthese purposes.

7.9 A Homolog of vAFP with Nucleotide Base Insertions

A homolog of variant AFP, characterized by insertion of T into position78 and C into position 123 and having a total length of 199 bases, anddescribed as SEQ ID NO:6, varies from variant A-AFP by two nucleotideinsertions. This form of variant A-AFP¹⁹⁹ and complements thereof andportions thereof can be used for identification of variant AFP in cellisolation—particularly in isolation of stem and progenitor cells frombone marrow and other hemopoietic organs, for cell cloning andidentification of cloned cells, for tumor identification, for evaluatingdevelopmental stages in organs and organisms, and as a hybridizationmarker for exon 1 or variants of exon 1. Other homologs of varA-AFP withabout 99% homology and having insertions are also useful for thesepurposes.

7.10 A Homolog of vAFP: A³⁰T⁵⁵C⁹⁰G¹⁰⁷T¹²⁴T¹³⁷-variant A-AFP¹⁹⁷

A homolog of variant AFP, designated A³⁰T⁵⁵C⁹⁰G¹⁰⁷T¹²⁴T¹³⁷-variantA-AFP¹⁹⁷, and described as SEQ ID NO:7, varies from variant A-AFP bysingle point substitutions at six loci. The variant A-AFP andcomplements thereof and portions thereof can be used for identificationof variant AFP in cell isolation—particularly in isolation of stem andprogenitor cells from bone marrow and other hemopoietic organs, for cellcloning and identification of cloned cells, for tumor identification,for evaluating developmental states in organs and organisms, and as ahybridization marker for exon 1 or variants of exon 1. Other homologs ofvarA-AFP with about 97% homology are also useful for these purposes.

7.11 Homologs of vAFP with about 90% Homology and about 80% Homology

Homologs of variant AFP, characterized by substitution of nucleotidebases such as to have about 90% homology and about 80% homology aredesignated SEQ ID NO:8 and SEQ ID NO:9, respectively. These forms ofvariant A-AFP¹⁹⁹ and complements thereof and portions thereof can beused for identification of variant AFP in cell isolation—particularly inisolation of stem and progenitor cells from bone marrow and otherhemopoietic organs, for cell cloning and identification of cloned cells,for tumor identification, for evaluating developmental stages in organsand organisms, and as a hybridization marker for exon 1 or variants ofexon 1. Other homologs of varA-AFP with about 90% and about 80% homologyare also useful for these purposes.

7.12 A Homolog of variantB-AFP: C⁷⁰C¹³⁰A¹⁸⁹-variantB-AFP³¹⁷

A homolog of variant AFP, designated C⁷⁰C¹³⁰A¹⁸⁹-variantB-AFP³¹⁷, anddescribed as SEQ ID NO:10, varies from variant B-AFP by single pointsubstitutions at three loci. The homolog and complements thereof andportions thereof can be used for identification of variant AFP in cellisolation—particularly in isolation of stem and progenitor cells frombone marrow and other hemopoietic organs, for cell cloning andidentification of cloned cells, for tumor identification, for evaluatingdevelopmental stages in organs and organisms, and as a hybridizationmarker for exon 1 or variants of exon 1. Other homologs of variantB-AFPwith about 99% homology are also useful for these purposes.

7.13 A Homolog of variantB-AFP: A⁵¹C¹⁷⁹T²³¹-variantB-AFP³¹⁷

A homolog of variant AFP, designated A⁵¹C¹⁷⁹T²³¹-variantB-AFP³¹⁷, anddescribed as SEQ ID NO:11, varies from variantB-AFP by single pointsubstitutions at three loci. The homolog and complements thereof andportions thereof can be used for all the purposes that variantB-AFP canbe used. Among suitable uses for the homolog can be (a) identificationof variant AFP in cell isolation—particularly in isolation of stem andprogenitor cells from bone marrow and other hemopoietic organs, (b) cellcloning and identification of cloned cells, (c) tumor identification,(d) evaluation of developmental stages in organs and organisms, and (e)as a hybridization marker for exon 1 or variants of exon 1. Otherhomologs of variantB-AFP with about 99% homology are also useful forthese purposes.

7.14 A Homolog of variantAFP:variantB-(des-C¹⁴⁰)(des-G¹⁸³)(des-C²²⁶)AFP³¹⁴

A homolog of variant AFP, designated variantB-(des-C¹⁴⁰)(des-G¹⁸³)(des-C²²⁶) AFP³¹⁴ and described as SEQ ID NO:12,varies from variant B-AFP by three nucleotide deletions. The homolog andcomplements thereof and portions thereof can be used for identificationof variant AFP in cell isolation—particularly in isolation of stem andprogenitor cells from bone marrow and other hemopoietic organs, for cellcloning and identification of cloned cells, for tumor identification,for evaluating developmental stages in organs and organisms, and as ahybridization marker for exon 1 or variants of exon 1. Other homologs ofvariantB-AFP with about 99% homology are also useful for these purposes.

7.15 A Homolog of variantB-AFP with Nucleotide Base Insertions

A homolog of variant B AFP, characterized by insertion of A intoposition 60, C into position 120, and T into position 295 and having atotal length of 320 bases, and described as SEQ ID NO:13, varies fromvariant B-AFP by three nucleotide insertions. This form of variantB-AFP³⁰⁰ and complements thereof and portions thereof can be used foridentification of variant AFP in cell isolation—particularly inisolation of stem and progenitor cells from bone marrow and otherhemopoietic organs, for cell cloning and identification of cloned cells,for tumor identification, for evaluating developmental stages in organsand organisms, and as a hybridization marker for exon 1 or variants ofexon 1. Other homologs of variantB-AFP with about 99% homology are alsouseful for these purposes.

7.16 A Homolog of variantB-AFP:A⁵¹A⁶⁷C⁷⁵T84G¹³⁸C¹⁷⁹T²³¹G²⁸⁹C²⁹²-variantB-AFP³¹⁷

A homolog of variant AFP, designatedA⁵¹A⁶⁷C⁷⁵T⁸⁴G¹³⁸C¹⁷⁹T²³¹G²⁸⁹C²⁹²-variantB-AFP³¹⁷ and described as SEQ IDNO:14, varies from variantB-AFP by single point substitutions at nineloci. The homolog and complements thereof and portions thereof can beused for all purposes that variantB-AFP can be used. Among suitableloses for the homolog can be (a) identification of variant AFP in cellisolation—particularly in isolation of stem and progenitor cells frombone marrow and other hemopoietic organs, (b) cell cloning andidentification of cloned cells, (c) tumor identification, (d) evaluationof developmental stages in organs and organisms, and (e) as ahybridization marker for exon 1 or variants of exon 1. Other homologs ofvariantB-AFP with about 97% homology are also useful for the purposes.

7.17 Homologs of variantB-AFP with about 90% Homology and about 80%Homology

Homologs of variant AFP, characterized by substitution of nucleotidebases such as to have about 90% homology and about 80% homology aredesignated SEQ ID NO:15 and SEQ ID NO:16, respectively. These forms ofvariant B-AFP³⁰⁰ and complements thereof and portions thereof can beused for identification of variant AFP in cell isolation—particularly inisolation of stem and progenitor cells from bone marrow and otherhemopoietic organs, for cell cloning and identification of cloned cells,for tumor identification, for evaluating developmental stages in organsand organisms, and as a hybridization marker for exon 1 or variants ofexon 1. Other homologs of variantB-AFP with about 90% or 80% homologyare also useful for these purposes.

7.18 Fluorescence In Situ Hybridization using Propidium IodideCounterstaining for Detection of Expression of vAFP mRNA

The present invention also encompasses in situ PCR and in situ RT-PCRfor detection of DNA and RNA related to normal and variant forms of AFP.The techniques are preferred when the copy number of a target nucleicacid is very low, or when different forms of nucleic acids must bedistinguished. The methods are especially important in detecting anddifferentiating precancer and cancer cells from normal cells. Themethods are also useful in detecting subsets of cells destined to becomecancer cells. Confirmation of in situ PCR product identity isaccomplished by in situ hybridization with a nested ³²P-labeled probe orby examining the products using Southern blot analysis to corroboratepredicted base pair size. Coordinate transcriptional/translationalexpression is demonstrated by sequential in situRT-PCR/immunohistochemical analysis on serial tissue sections.

Fluorescence in situ hybridization (FISH) in combination with propidiumiodide (PI) counterstaining is used to demonstrate mRNA expression ofauthentic and variant AFP in tissue sections. One suitable generalmethod is described by Wulf, M., et al. Biotechniques, Vol. 19, No. 3,pp. 368-372, 1995.

After surgical removal, tissue samples are immediately fixed in 10%formaldehyde (pH 7.0) and nondecalcified, paraffin-embedded specimensare used for FISH. Pretreatment of sections before hybridization iscarried out by covering the sections with 300 .mu.l of prehybridizationbuffer (50% deionized formamide, 0.3 M NaCl, 10 mM Tris-HCl, pH 7.5; 10mM NaHPO.sub.4, pH 6.8; 5 mM EDTA; 0.1.times. Denhardt's, 10 mMdithiothreitol; 0.25 mg/ml yeast tRNA, 12.5% dextran sulfate; 0.5 mg/mlsalmon sperm DNA and is incubated in a humid chamber for 2 hr at42.degree. C. For hybridization, digoxigenin-labeled double-strandedcDNA probe for the vAFP having the sequence 5-ACCATGAAGTGGGTGGAATC-3′(SEQ ID NO:41) (ex-1S, Table 1) and 5′-ATTTAAACTCCCAAAGCAGCAC-3′ (SEQ IDNO:49) (ex-14A, Table 1) are used. The probe is labeled with digoxigeninaccording to the protocol of the Dig-Labeling Kit (Boehringer Mannheim,Mannheim, Germany). Prior to hybridization, the labeled probe is mixedwith prehybridization buffer to a concentration of 1 .mu.g/mL, heatedfor 10 mm. at 95.degree. C. and quickly chilled on ice. Excessprehybridization buffer is removed from the slides, and approximately 30.mu.l of hybridization solution is applied to the sections. Sections arecovered with a coverslip, sealed with rubber cement and hybridized in ahumid chamber at 42.degree. C. for 18 h. The post-hybridization washingsteps are performed as described by Weithege, T., et al. Pathol. Res.Pract., 187:912-915, 1991.

Probe detection is carried-out using an anti-digoxigenin antibodyconjugated to FITC (fluorescein isothiocyanate). Unbound conjugate isremoved by washing two times for 10 min. with phosphate-buffered saline(PBS) (3.8 mM NaH₂ PO₄; 7.8 mM Na₂ HPO₄; 0.13 M NaCl). Sections arecounterstained with PI in PBS (500 ng/mL) for 5 min. at room temperature(30 μl per section). Excess PI is removed by washing with PBS, followedby dehydration (70%, 96%, 100% ethanol). Sections are air-dried andmounted in a glycerol/PBS solution. For analyses, a fluorescencemicroscope is used.

Using FISH, differential expression of the authentic AFP or vAFP mRNA inprecancer and cancer cells is determined as compared to normal cells.

7.19 In Situ PCR and In Situ RT-PCR of Paraffin-Embedded Tissue Sectionsfor Localization of Nucleic Acids of vAFP

The following protocol is used to detect nucleic acids of AFP and vAFPwhich may be associated with precancer and cancer, in precancer andcancer cells. The method is also useful to detect the chromosomallocation of the nucleic acid or chromosomal abnormalities at thelocation

Cell Lines

HepG2 and K562 cell lines are used in this study. Pellets ofapproximately 5×10⁶ cells are washed in PBS, re-suspended in 1 ml of 2%NuSieve low melting-point agarose allowed to solidify, fixed for 2 hr in4% paraformaldehyde or 10% formalin, and embedded in paraffin by routinehistopathology techniques.

RNA Extraction

The guanidine isothiocyanate-cesium chloride method of Glisin et al(Biochemistry Vol. 13; 2633, 1974) is used to extract total RNA from thecell lines. Poly A+ RNA from normal human brain, liver, thymus, stomach,and bone marrow are used.

Northern Blot

Standard formaldehyde gels were run with total RNA (10 μg/well) at 120v. 100 mAmp for 3 hr. At the end of the run, the gels are washed for 15min in 20×. SSC and then blotted overnight by capillary flow transferonto a 0.45-.μ.m nitrocellulose filter. The blots are UV crosslinked at1200 Joules and pre-hybridized for 4 hr. The Stratagene Prime-It kit(Stratagene; La Jolla, Calif.) is used to label the probe. The probesare prepared by random priming of inserts gel purified from restrictionendonuclease digests of plasmids containing full-length cDNAs for vAFPwith ³²P-dCTP. Probe (1×10⁶ cpm) is added to each ml of hybridizingbuffer. After overnight hybridization, the blot is treated under thefollowing stringent conditions: washed once in 2×SSC/0.1% SDS at roomtemperature, the blot is washed once in 2×SSC/0.1% at room temperature(RT; 30 min) and once with 0.1% SSC, 0.1% SDS at 60C. (30 min). Theblots are then air-dried and autoradiographed at −80° C. on Kodak XAR5film for 1-2 days.

Standard PCR

Oligonucleotide primers for vAFP are made using a MilliGen 8700 DNAsynthesizer. Sequences are 5′-CTTCCATATTGGATTCTTACCCAATG-3′ (SEQ IDNO:66) (ex-2S, Table 1) and 5′-TAAACCCTGGTGTTGGCCAG-3′ (SEQ ID NO:47)(ex-125, Table 1). All buffers, enzymes, and nucleotides used areobtained from Applied Biosystems. PCR products are analyzedelectrophoretically using a 1% agarose gel (80 V, 3 hr) and the ethidiumbromide staining is observed under UV light, followed by Southernanalysis with nested .sup.32P-labeled probes.

Southern Analysis

Gels are denatured in 1.5 M NaCl/0.6 M NaOH and 1.5 M NaCl/2 M Tris andblotted onto a 0.2-μm nitrocellulose filter in 20×SSC by capillary flowtransfer overnight. The filters are cross-linked at 80° C. under vacuumand put in hybridization buffer. Anti-sense nested probes areend-labeled by standard ³²P procedures. Hybridization with the probe isdone overnight at 42° C. Stringency washing at RT is in 5×SSC/0.1% SDS(twice for 30 min), then 1×SSC/0.1% SDS (twice for 30 min). Filters areair-dried and autoradiographed at −80° C. on Kodak XAR5 film for 2-4 hr.

In Situ PCR

The in situ PCR technique for localizing specific DNA sequences isperformed by a three-step protocol. After dewaxing the tissue sections,a protein digestion is carried out to facilitate reagent penetrationinto the cells. A second step consists of the PCR itself withsimultaneous labeling of the PCR products, followed by a third step thatvisualizes the labeled product. The in situ amplification technique forRNA detection utilizes a similar protocol. However, it incorporates twoadditional steps. After proteinase digestion the tissue is exposed toRNAse-free DNAse to avoid amplification of genomic DNA. Second, theremaining mRNA is reverse-transcribed to form cDNA templates, which arein turn amplified by PCR. To maximize the efficiency of the in situ PCRtechnique, all of these protocol steps must be optimized for individualanalysis. The reverse transcription and the PCR steps is performed usingan OmniSlide thermocycler (20-slide capacity) equipped with a heatedwash module.

Protease Digestion

Depending on the fixative and the nature of the tissue, reagent accessto the target nucleic acid can vary. Optimal permeability methods, canbe obtained by varying the concentration of proteinase K between 1 and100 μg/ml and incubation time (5-45 min).

DNAse Digestion

Deoxyribonuclease I Amplification Grade 10 U/slide is used to degradethe DNA according to standard methods. The influence of differentdigestion times on the quality of the staining is tested.

Reverse Transcription

For this step the SuperScript Preamplification System is used followingstandard methods. In summary, the sections are immersed in a solutioncontaining the random primers, covered with parafilm coverslips, andincubated in the thermocycler for 10 min at 70° C. After removing thecoverslips, another solution containing the reverse transcriptase (100U/section) is added and covered with a new piece of parafilm. The slidesare then maintained at RT for 10 min, at 45° C. for 45 min, and at 70°C. for 10 min.

PCR

Before the in situ PCR experiment, all parameters for the PCR reaction,including MgCl₂ concentration, pH, and annealing temperature, areoptimized by standard PCR. At this point the PCR products can be clonedand sequenced to confirm identity. Optimization of conditions favoringsingle band production is advised because it is not possible todistinguish PCR products of different molecular weights in the tissuesections. To eliminate the possibility of generating PCR products fromgenomic DNA, it is important to design primers that bridge introns so asto distinguish template source on the basis of product size.

Synchronized “hot start” PCR is achieved using the Taq neutralizingantibody technique (Kellogg et al., Bio Techniques 6:1134, 1994).

The following PCR mixture is used: 2.5 μM MgCl₂ 200 μM dNTP2, 100 μMdigoxigenin-11-2′-deoxyuridine-5′-triphosphate, 1 ng/μl primers, 50 μMKCl, 10 μM Tris-HCL, pH 8.3. An 80-μl aliquot of solution is applied toeach slide, and then each slide is covered by silanated glasscoverslips, sealed with rubber cement, and placed in the thermocycler.The targets are amplified, 15-20 cycles to obtain crisp staining. AfterDNA amplification, two washes in 0.1×SSC at 45° C., 20 min each, areperformed to eliminate unbound nucleotides.

Development of Digoxigenin

Detection of digoxigenin-tagged PCR products can be done with a kitstandard in the art. It involves a 2-hr incubation with ananti-digoxigenin antibody bound to alkaline phosphatase. After athorough rinse, the appropriate substrates (nitroblue tetrazolium and5-bromo-chloro-3-indolyl-phosphate) are enzymatically transformed into adark blue precipitate. Color deposition is checked under the microscope.

Polyvinyl alcohol can enhance the intensity of the alkalinephosphatase-nitroblue tetrazolium reaction and prevent diffusion of theprecipitate. To take advantage of this technique the dilution of theanti-digoxigenin antibody is increased to 1:2000 (instead of the usual1:500 recommended by the manufacturer) to obtain considerable backgroundreduction.

Controls

The PCR technique is well known for its ability to amplify even singlecopies of DNA in a sample, contaminants included. Therefore, precautionsthat are recommended for routine PCR with regard to scrupulous care withcleanliness, use of a dedicated set of pipettes, and preparation of thePCR mixture away from the amplification area are also applicable for insitu PCR. In addition, working with tissue sections adds new concerns,such as heterogeneous application of reagents, bubbles, drying of theboundaries, and stability of the nucleic acids during the preparation ofthe samples.

At least three types of controls are recommended in every experiment toavoid false-positives or -negatives.

Positive Control

Include a section from a block that has previously been found positivefor the same set of primers. If this is the first time that theseprimers are being used, a section of a well-fixed tissue or cell lineknown to have a high expression of the target nucleic acid as determinedby other techniques (e.g., Northern analysis, standard PCR, in situhybridization is included).

Negative Controls

Omission of the reverse transcription and/or RNAse treatment will yieldinformation about nonspecific amplification of remaining nuclear ormitochondrial DNA.

Omission of the primers in the PCR mixture will reveal nonspecificstaining due to endogenous priming: DNA fragments produced by theexonuclease activity of the DNA polymerase and other artifacts such asintrinsic alkaline phosphatase activity.

An additional control consists of establishing existing relationshipbetween the transcriptional/translational products. This can be done bystaining one section for the nucleic acid by in situ PCR and a serialsection with a specific antibody against the polypeptide. Theco-localization of the mRNA and its protein within the same cells willstrengthen the validity of the observation, can be applied to authenticAFP.

Confirmation of the in situ PCR product integrity can be achieved in twoways: (a) It is possible to scrape the tissue of the glass slide afterin situ PCR, to extract the DNA and to analyze by agarose gelelectrophoresis and Southern blot with the appropriate radioactiveprobe. Cloning and sequencing of this product is also possible, afterseveral additional PCR cycles to yield products without modified bases,(b) Product identity is tested by performing in situ hybridization witha ³²P-labeled nested probe after the amplification. This procedure canbe used for indirect in situ PCR.

7.20 Variant AFP Expression in Fetal Tissues

The expression of vAFP by in situ hybridization in fetal tissue isevaluated to determine if these molecules were potentially involved inearly organogenesis. This would establish vAFP as an oncofetal antigenand provide additional support for the hypothesis that vAFP isindicative of the process of carcinogenesis and fetal development.Multiple sections of human tissue from various stages of embryonaldevelopment and adult are evaluated.

Sections (4 μm thick) are mounted on slides coated with Vectabond,dewaxed and prepared for hybridization with RNA probes. In summary, thevAFP DNA fragment can be subcloned into a suitable vector and linearizedwith the appropriate restriction enzymes. Labeled probes are preparedusing digoxigenin-11-UTP and T7 or T3 RNA polymerases to synthesizesense and antisense RNA transcripts, respectively. Hybridization isperformed in a moist chamber at 46° C. for 20 hours in a 15-μl volumecontaining 0.5 ng/μl of probe for each section. Stringency washesincluded treatments with 150 mmol/L NaCl, 15 mmol/L sodium citrate, pH7.0 (SSC), and sodium dodecyl sulfate (SDS) as follows: four washes in2×SSC/0/1% SDS, two washes in 0.1×SSC/0.1% SDS at 46° C., brief rinsesin 2×SSC, incubation in 2×SSC containing 10 μg/ml RNAse at 37° C. for 15minutes, and additional rinses in 2×SSC.

Visualization of digoxigenin is performed with a monoclonal antibodycoupled to alkaline phosphatase diluted about 1:500 acting for 2 hoursat room temperature. Nitroblue tetrazolium chloride and5-bromo-4-chloro-3-indoly-phosphate are used as substrates for thealkaline phosphatase. The use of the sense probe and treatment of thesections with RNAse before the hybridization are included.

7.21 Interpretation and Mechanistic Underpinnings of the Invention

The present invention identifies at least two variant forms of AFPexpressed preferentially in hemopoietic progenitors, but not in matureblood cells or in hepatic cells. The invention is not limited by themechanisms discussed. These results suggest that the AFP gene locus ofchromatin in hemopoietic progenitor is open and accessible totranscription factors for the mRNA expression. In other words,chromatin-related repression of authentic AFP, which is a mechanism toblock inappropriate expression of authentic AFP in non-endodermal cells,is incomplete in hemopoietic progenitors and allows vAFP to betranscribed. In the case of K562, strictly speaking, very small amountsof authentic AFP transcripts could be detected when the PCR cycles wereincreased (FIG. 5B). This indicates that K562 express vAFP at muchhigher level than authentic AFP. Conversely, vAFP could be detected inHepG2 by increasing cycles of RT-PCR. Therefore, our data taken from thecell lines suggest that three types of patterns are present with respectto the authentic and variant transcripts of AFP. Hepatic cells (HepG2)express authentic AFP dominantly, while hemopoietic cells (K562) expressvAFP dominantly. Fibroblasts (MRC5) express neither authentic AFP norvAFP. These results prompt two questions with respect to AFP expression.One is what is the mechanism of the opening of the AFP locus in thechromatin giving rise to whichever of the transcripts is expressed. Theother is what is the transcriptional machinery associated with thedifferent forms of transcripts.

The different usage of the first exon suggests the vAFP expression isdependent on the unique promotor. The mouse has proved an excellentmodel system for studying tissue-specific and developmentally regulatedtranscriptional control of authentic AFP in vivo as well as in vitro.Extensive studies have established that the transcriptional control ofthe AFP gene is mediated by five cis-acting regulatory domains,including the AFP promoter, three distinct enhancer elements and onerepressor region located between the AFP promoter and the upstreamenhancers. There are a number of transcription factors binding in thepromotor region and involving the expression of the authentic AFP form.GATA4 could be a master gene for AFP gene expression and endodermaldifferentiation. In addition, GATA1 and GATA2 are indispensable factorsfor early hemopoietic differentiation. Interestingly, several possibleGATA family binding sites are identified in the exon A genomic sequenceas well as other transcription factor binding sites associated withhematopoiesis such as MZF-1 and AML-1. Thus, it is noteworthy to seewhether these transcription factors expressed in hemopoietic progenitorsare involved in the transcription of vAFP. Although there have beenreports on the different promoters of the variant AFP transcripts inmouse and rat, there is no report about human variant AFP transcriptsnor identification of exon A and exon B at 5′ upstream of exon 1 in anyspecies.

There is an explosion of studies about the possibility oftransdifferentiation events in mammals. While evidence for thephenomenology is expanding rapidly, almost all of the evidence isinconclusive, and the approach to elucidate possible mechanism(s) isvery limited. As a new model system, the AFP gene expression is ideal,because the gene is one of the most characterized in vivo and in vitrowith respect to cell type-specific expression. A dynamic transition ofthe gene expression pattern could be measured by using gene array orgene tip technology if an in vitro conditional system with respect tothe expression of different forms of AFP is developed. This approachcould provide proof of transdifferentiation between hepatic andhemopoietic cells and be suggestive of a possible mechanism(s).

At present, apart from hemopoietic organs, brain is the only tissue inwhich vAFP expression is observed. Although there are claims that neuralstem cells in the subventricular zone (SVZ) of the brain are able todifferentiate to endodermal cells, the data are not yet conclusive.Therefore, the finding of vAFP+ cells in the brain is highly interestingand suggests, at the least, that cells in the brain possibly progenitorsmay share aspects of the developmental potential of endodermalprogenitors just as CD34+Lin− hemopoietic progenitors do.

The function of the vAFP is unclear, because the amount of mRNA isextremely low and no protein products are found in assays usingimmuno-histochemistry with anti-AFP antibodies. In addition, there areno long open reading frame starts in exon A or B to connect exon 2 ofauthentic AFP if only ATG is considered as an initiation codon. In thatcase, an initiation codon at exon 3 would be used for the translation sothat the translated product from vAFP transcripts would be a truncatedform. However, since there are TTG and CTG in exon B to connect the openreading frame of exon 2, one of them could work as an alternativeinitiation codon.

TABLE 1 Primer sequences used in this study (SEQ ID NOS: 41-65 disclosedrespectively in order of appearance) RT-PCR ex-1S5′-ACCATGAAGTGGGTGGAATC-3′ ex-2S 5′-CTTCCATATTGGATTCTTACCAATG-3′ ex-3S5′-GGCTACCATATTTTTTGCCCAG-3′ ex-4S 5′-CTACCTGCCTTTCTGGAAGAAC-3′ ex-5S5′-GAGATAGCAAGAAGGCATCCC-3′ ex-6S 5′-AAAGAATTAAGAGAAAGCAGCTTG-3′ ex-12S5′-TAAACCCTGGTGTTGGCCAG-3′ ex-3A 5′-CCTGAAGACTGTTCATCTCC-3′ ex-14A5′-ATTTAAACTCCCAAAGCAGCAC-3′ ex-AS 5′-AGAATTAAGGGACAGACTATGGGC-3′ ex-A1S5′-GATGTCTGTCTCATAACACTTGGG-3′ ex-A2S 5′-TAAGCTTGGCAACTTGCAACAGGG-3′ex-1capS 5′-ATATTGTGCTTCCACCACTGCC-3′ beta-actin S5′-TGCAAGGCCGGCTTCGCGGGC-3′ beta-actin A 5′-TCCTTCTGCATCCTGTCGGCA-3′CD34S 5′-TCATGAGTCTTGACAACAACGG-3′ CD34A 5′-CAGCCACCACGTGTTGTCTTGC-3′1.0. Anchored PCR SN-poly(C) 5′-ACTAGTTAGCGGCCGCACTGGGC¹⁴-3′ SN-primer5′-ACTAGTTAGCGGCCGCACTGGG-3′ ex-1A 5′-CTCACCTATTCCATATTCATTTC-3′ ex-2A5′-GGTCAGCTAAACTTATCTCTGC-3′ ex-3A 5′-GGCTTCTTGAACAAACTGGGC-3′ ex-4A5′-GCTGCAGCAGTCTGAATGTCC-3′ 2.0. Genomic cloning of exonA g-ex-AS5′-ATTCTGTTTTCACCCCATAGGTG-3′ g-ex-AA 5′-TTTCTCAGATATTCAAGCCCCAG-3′

1. An isolated nucleic acid encoding a variant form of humanalpha-fetoprotein, in which exon 1 of exons 1-14 of cDNA encodingalpha-fetoprotein has been replaced by the polynucleotide sequence ofexon A (SEQ ID NO:26), or a fusion of exon A and exon B (SEQ ID NO:27).2. The nucleic acid of claim 1 in which the nucleic acid encoding thevariant form of human alpha-fetoprotein includes at least 100 contiguousnucleotides of the polynucleotide sequence of exon A, or a fusion ofexon A and exon B encoding the N-terminus of the variant form ofalpha-fetoprotein.
 3. The nucleic acid of claim 1 in which the nucleicacid encoding the variant form of human alpha-fetoprotein includes atleast 100 contiguous nucleotides of the polynucleotide sequence of exonA, or a fusion of exon A and exon B encoding the N-terminus of thevariant form of alpha-fetoprotein.